Glucagon-like peptide-1 crystals

ABSTRACT

The invention provides individual tetragonal flat rod shaped or plate-like crystals of glucagon-like peptide-1 related molecules, processes for their preparation, compositions and methods of use. The crystal preparations exhibit extended time action in vivo and are useful for treating diabetes, obesity and related conditions.

This application is a continuation of U.S. Ser. No. 09/209,799, filedDec. 11, 1998, now U.S. Pat. No. 6,380,357, which claims the benefit ofU.S. Provisional Application Serial No. 60/069,728, filed Dec. 16, 1997.

FIELD OF THE INVENTION

The present invention relates to peptide chemistry as it applies topharmaceutical research and development. The invention providesindividual tetragonal flat rod shaped or plate-like crystals ofglucagon-like peptide-1 related molecules, processes for theirpreparation, compositions and uses for these improved crystal forms.

BACKGROUND OF THE INVENTION

GLF-1, a 37 amino acid peptide naturally formed by proteolysis of the160 amino acid precursor protein preproglucagon, was first identified in1987 as an incretin hormone. GLP-1 is secreted by the L-cells of theintestine in response to food ingestion and has been found to stimulateinsulin secretion (insulinotropic action) causing glucose uptake bycells which decreases serum glucose levels (see, e g., Mojsov, S., Int.J. Peptide Protein Research, 40:333-343 (1992)). GLP-1 is poorly active.A subsequent endogenous cleavage between the 6^(th) and 7^(th) positionproduces a more potent biologically active GLP-1(7-37)OH peptide.Approximately 80% of the GLP-1(7-37)OH so produced is amidated at theC-terminal in conjunction with removal of he terminal glycine residuesin the L-cells and is commonly referred to GLP-1(7-36)NH₂. Moleculeswhich are reasonably homologous to, or are derived from, or based onthese native forms will generally be referred to as GLP's in thisspecification.

The biological effects and metabolic turnover of the free acid, theamide form, and many of the numerous known GLP's are similar and showpromise as agents for the treatment of diabetes, obesity, and relatedconditions, including but not limited to impaired glucose tolerance andinsulin resistance. However, many GLP's suffer from extremely shortbiological half lives, some as short as 3-5 minutes, which makes themunattractive for use as pharmaceutical agents. Presently, the activityof dipeptidyl-peptidase-IV (DPP-IV) is believed to readily inactivatemany GLP's and is in part responsible for the very short serum halflives observed. Rapid absorption and clearance following parenteraladministration are also factors. Thus, there is a need to find a meansfor prolonging the action of these promising agents.

One such approach has been to modify these molecules to protect themfrom in vivo cleavage by DPP-IV. For example, see U.S. Pat. No.5,512,549. In the insulin arts, it has long been known that extendedtime action can be achieved by administering crystalline proteinformulations into the subcutis which act like depots, paying out solubleprotein over time.

Heterogeneous micro crystalline clusters of GLP-1(7-37)OH have beengrown from saline solutions and examined after crystal soaking treatmentwith zinc and/or m-cresol (Kim and Haren, Pharma. Res. Vol. 12 No. 11(1995)). Also, crude crystalline suspensions of GLP(7-36)NH₂ containingneedle-like crystals and amorphous precipitation have been prepared fromphosphate solutions containing zinc or protamine (Pridal, et. al.,International Journal of Pharmaceutics Vol. 136, pp. 53-59 (1996)).Also, EP 0 619 322 A 2 describes the preparation of micro-crystallineforms of GLP-1(7-37)OH by mixing solutions of the protein in pH 7-8.5buffer with certain combinations of salts and low molecular weightpolyethylene glycols (PEG). However, such crystalline clusters and crudesuspensions are less than ideal for preparing long acting pharmaceuticalformulations of GLP's since they are loosely bound heterogeneousclusters of crystals or amorphous-crystalline suspensions which tend totrap impurities and are otherwise difficult to reproducibly manufactureand administer.

Most unexpectedly it was discovered that single tetragonal flat rodshaped or plate-like crystals Of various GLP's could be reproduciblyformed from a mother liquor containing a GLP dissolved in a bufferedsolution and a C₁₋₃ alcohol, or optionally a mono or disaccharide, overa wide range of pH conditions. The resulting single flat rod shaped orplate-like crystals are superior to, and offer significant advantagesover, the GLP-1(7-37)OH crystal clusters or crude suspensions known inthe art.

The single tetragonal flat rod shaped or plate-like crystals of thepresent invention are less prone to trap impurities and therefore may beproduced to greater yields and administered more reproducibly than theknown heterogeneous clusters. The crystal compositions of the presentinvention are pharmaceutically attractive because they are relativelyuniform and remain in suspension for a longer period of time than thecrystalline clusters or s amorphous crystalline suspensions which tendto settle rapidly, aggregate or clump together, clog syringe needles andgenerally exacerbate unpredictable dosing. Most importantly, the crystalcompositions of the present invention display extended, uniform, andreproducible pharmacokinetics which can be modulated by adding zincusing conventional crystal soaking techniques or, alternatively, byincluding zinc in the crystallization solution.

BRIEF SUMMARY OF THE INVENTION

The present invention includes processor for preparing single rod-shapedor plate-like crystals of glucagon-like peptide-1 related molecules(GLP's) which comprises preparing a crystallization solution comprisinga purified GLP, a buffering agent containing an alcohol or a mono or disaccharide, and optionally, ammonium sulfate or zinc. In anotherembodiment the GLP crystals having tetragonal flat rod shaped orplate-like morphology selected from the group consisting of a GLP-1analog, a GLP-1 derivative, a DPP-IV protected GLP, a GLP-1 peptideanalog, or a biosynthetic GLP-1 analog are claimed. The invention alsoincludes substantially homogenous compositions of GLP crystals,pharmaceutical formulations and processes for preparing suchformulations, and methods for treating diabetes, obesity and relatedconditions.

DETAILED DESCRIPTION OF THE INVENTION

By custom in the art, the amino terminus of GLP-1(7-37)OH has beenassigned number residue 7 and the carboxy-terminus, number 37. Thisnomenclature carries over o other GLP's. When not specified, theC-terminal is usually considered to be in the traditional carboxyl from.The amino acid sequence and preparation of GLP-1(7-37)OH is well-knownin the art. See U.S. Pat. No. 5,120,712, the teachings of which areherein incorporated by reference. For the convenience of the reader thesequence is provided below.

-   -   His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-Gly-COOH        (SEQ ID NO:1)

“GLP-1 analog” is defined as a molecule having one or more amino acidsubstitutions, deletions, inversions, or additions relative toGLP-1(7-37) and may include the d-amino acid forms. Numerous GLP-1analogs are known in the art and include, but are not limited to,GLP-1(7-34) (SEQ ID NO:6), GLP-1(7-35) (SEQ ID NO:7), GLP-1(7-36)NH₂(SEQ ID NO:8), Gln⁹-GLP-1(7-37) (SEQ ID NO: 9), d-Gln⁹-GLP-1(7-37) (SEQID NO:10), Thr¹⁶-Lys¹⁸-GLP-1(7-37) (SEQ ID NO:11), and Lys¹⁸-GLP-1(7-37)(SEQ ID NO:12), Gly⁸-GLP-1(7-36)NH₂ (SEQ ID NO:13), Gly⁸-GLP-1(7-37)OH(SEQ ID NO: 14), Val⁸-GLP-1(7-37)OH (SEQ ID NO:5), Met⁸-GLP-1(7-37)OH(SEQ ID NO:15), acetyl-Lys⁹-GLP-1(7-37) (SEQ ID NO:16), Thr⁹-GLP-1(7-37)(SEQ ID NO:17), d-Thr⁹-GLP-1(7-37) (SEQ ID NO:18), Asn⁹-GLP-1(7-37) (SEQID NO:19), d-Asn⁹-GLP-1(7-37)(SEQ ID NO:20), Ser²²-Arg²³Arg²⁴-Gln²⁶-GLP-1(7-37)(SEQ ID NO:21), Arg²³-GLP-1(7-37) (SEQ ID NO:22),Arg²⁴-GLP-1(7-37) (SEQ ID NO:23), α-methyl-Ala⁸-GLP-1(7-36)NH₂ (SEQ IDNO:24), and Gly⁸-Glu²¹-GLP-1(7-37)OH (SEQ ID NO:25), and the like.

Other GLP-1 analogs consistent with the present invention are describedby the formula:

-   -   R₁-X-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Y-Gly-Gln-Ala-Ala-Lys-Z-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-R₂        (SEQ ID NO:2)        wherein: R₁ is selected from the group consisting of        L-histidine, D-histidine, desamino-histidine, 2-amino-histidine,        beta-hydroxy-histidine, homohistidine,        alpha-fluoromethyl-histidine, and alpha-methyl-histidine; X is        selected from the group consisting of Ala, Gly, Val, Thr, Met,        Ile, and alpha-methyl-Ala; Y is selected from the group        consisting of Glu, Gln, Ala, Thr, Ser, and Gly; Z is selected        from the group consisting of Glu, Gln, Ala, Thr, Ser, and Gly;        and R₂ is selected from the group consisting of NH₂, and Gly-OH.

GLP-1 analogs have also been described in WO 91/11457, and includeGLP-1(7-34), GLP-1(7-35), GLP-1(7-36), or GLP-1(7-37), or the amide formthereof, and pharmaceutically-acceptable salts thereof, having at leastis one modification selected from the group consisting of:

-   -   (a) substitution of glycine, serine, cysteine, threonine,        asparagine, glutamine, tyrosine, alanine, valine, isoleucine,        leucine, methionine, phenylalanine, arginine, or D-lysine for        lysine at position 26 and/or position 34; or substitution of        glycine, serine, cysteine, threonine, asparagine, glutamine,        tyrosine, alanine, valine, isoleucine, leucine, methionine,        phenylalanine, lysine, or a D-arginine for arginine at position        36;    -   (b) substitution of an oxidation-resistant amino acid for        tryptophan at position 31;    -   (c) substitution of at least one of: tyrosine for valine at        position 16; lysine for serine at position 18; aspartic acid for        glutamic acid at position 21; serine for glycine at position 22;        arginine for glutamine at position 23; arginine for alanine at        position 24; and glutamine for lysine at position 26; and    -   (d) substitution of at least one of: glycine, serine, or        cysteine for alanine at position 8; aspartic acid, glycine,        serine, cysteine, threonine, asparagine, glutamine, tyrosine,        alanine, valine, isoleucine, leucine, methionine, or        phenylalanine for glutamic acid at position 9; serine, cysteine,        threonine, asparagine, glutamine, tyrosine, alanine, valine,        isoleucine, leucine, methionine, or phenylalanine for glycine at        position 10; and glutamic acid for aspartic acid at position 15;        and    -   (e) substitution of glycine, serine, cysteine, threonine,        asparagine, glutamine, tyrosine, alanine, valine, isoleucine,        leucine, methionine, or phenylalanine, or The D- or N-acylated        or alkylated form of histidine for histidine at position 7;        wherein, in the substitutions in (a), (b), (d), and (e), the        substituted amino acids can optionally be in the D-form and the        amino acids substituted at position 7 can optionally be in the        N-acylated or N-alkylated form.

A “GLP-1 derivative” is defined as a molecule having the amino acidsequence of GLP-1(7-37) or of a GLP-1 analog, but additionally havingchemical modification of one or more of its amino acid side groups,α-carbon atoms, terminal amino group, or terminal carboxylic acid group.A chemical modification includes, but is not limited to, adding chemicalmoieties, creating new bonds, and removing chemical moieties.Modifications at amino acid side groups include, without limitation,acylation of lysine ε-amino groups, N-alkylation of arginine, histidine,or lysine, alkylation of glutamic or aspartic carboxylic acid groups,and deamidation of glutamine or asparagine. Modifications of theterminal amino include, without limitation, the desamino, N-lower alkyl,N-di-lower alkyl, and N-acyl modifications. Modifications of theterminal carboxy group include, without limitation, the amide, loweralkyl amide, dialkyl amide, and lower alkyl ester modifications. Loweralkyl is C₁-C₄ alkyl. Furthermore, one or more side groups, or terminalgroups, may be protected by protective groups known to theordinarily-skilled protein chemist. The α-carbon of an amino acid may bemono- or dimethylated.

Other GLP-1 derivatives are claimed in U.S. Pat. No. 5,188,666, which isexpressly incorporated by reference. Such molecules are selected fromthe group consisting of a peptide having the amino acid sequence:

-   -   His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-X        (SEQ ID NO:3)        and pharmaceutically-acceptable salts thereof, wherein X is        selected from the group consisting of Lys-COOH and Lys-Gly-COOH;        and a derivative of said peptide, wherein said peptide is        selected from the group consisting of: a        pharmaceutically-acceptable lower alkyl ester of said peptide;        and a pharmaceutically-acceptable amide of said peptide selected        from the group consisting of amide, lower alkyl amide, and lower        dialkyl amide.

Yet another GLP-1 derivatives consistent for use in the presentinvention include compounds claimed in U.S. Pat. No. 5,512,549, which isexpressly incorporated herein by reference described by the formula:

wherein R¹ is selected from the group consisting of 4-imidazopropionyl,4-imidazoacetyl, or 4-imidazo-α, α dimethyl-acetyl; R² is selected fromthe group consisting of C₆-C₁₀ unbranched acyl, or is absent; R³ isselected from the group consisting of Gly-OH or NH₂; and, Xaa is Lys orArg, may be used in present invention.

“DPP-IV protected GLP's” refers to GLP-1 analogs which are resistant tothe action of DPP-IV. These include analogs having a modified or d aminoacid residue in position 8. These also include biosynthetic GLP-1analogs having Gly or the 1 amino acid residues Val, Thr, Met, Ser, Cys,or Asp in position 8. Other DPP-IV protected GLP's include des aminoHis⁷ derivatives.

“GLP-1 peptide analogs” are defined as GLP-1 analogs or derivativeswhich exclude acylated forms.

“Biosynthetic GLP-1 analogs” are defined as any GLP-1 analogs orderivatives which contain only naturally occurring amino acid residuesand are thus capable of being expressed by living cells, includingrecombinant cells and organisms.

“Treating” is defined as the management and care of a patient for thepurpose of combating the disease, condition, or disorder and includesthe administration of a compound of present invention to prevent theonset of the symptoms or complications, alleviating the symptoms orcomplications, or eliminating the disease, condition, or disorder.Treating diabetes therefore includes the maintenance of physiologicallydesirable blood glucose levels in patients in need thereof.

The flat rod shaped or plate-like GLP crystals of the present invention,which are prepared using the claimed process, vary in size and shape tosome degree. Generally, they range in size from approximately 2-25microns (μm) by 10-150 μm and are flat, having a depth of approximately0.5-5 μm. These single crystals form from a single nucleation point anddo not appear as multiple spiked star-like clusters known in the art.

Given the sequence information herein disclosed and the state of the artis solid phase protein synthesis, GLP's can be obtained via chemicalsynthesis. However, it also is possible to obtain some GLP's byenzymatically fragmenting proglucagon mating techniques well known tothe artisan. Moreover, well known recombinant DAN techniques may be usedto express GLP's consistent with the invention.

The principles of solid phase chemical synthesis of polypeptides arewell known to the art and may be found in general texts in the area suchas Dugas, H. and Penney, C., Bioorganic Chemistry (1981)Springer-Verlag, New York, pgs. 54-92, Merrifield, J. M., Chem. Soc.,85:2149 (1962), and Stewart and Young, Solid Phase Peptide Synthesis,pp. 24-66, Freeman (San Francisco, 1969).

Likewise, the state of the art in molecular biology provides theordinarily skilled artisan another means by which GLP's can be obtained.Although GLP's may be produced by solid phase peptide synthesis,recombinant methods, or by fragmenting glucagon, recombinant methods arepreferable when producing biosynthetic GLP-1 analogs because higheryields are possible.

For purposes of the present invention, GLP-1 peptide analogs andbiosynthetic GLP-1 peptide analogs are preferred. More preferred are theDPP-IV protected GLP's, More highly preferred are biosynthetic GLP-1peptide analogs. Another preferred group of GLP-1 peptide analogs arethose which contain a single amino acid substitution at the 8 positionwhich may include d and modified amino acid residues. More highlypreferred biosynthetic GLP-1 peptide analogs are those which contain asingle amino acid substitution at the 8 position, more preferably thosewhich contain Gly or the 1 amino acid residues Val, Thr or Met in the 8position.

The present invention provides a process for producing individualtetragonal rod shaped GLP crystals from a mother liquor. Under low toneutral pH conditions ranging from about pH 6-7, preferably about6.4±about 0.2, the crystallization solution, or mother liquor, containsa final GLP concentration of about 1-10 mg/ml, preferably 2-7 mg/ml.

A number of conventional buffer solutions containing an alcohol or monoor disaccharide are suitable in the practice of the invention. 10 to 50mM Tris, ammonium acetate, sodium acetate, or Bis-Tris is preferred. Theconcentration of alcohol ranges from about 2-15% (v/v), preferably3-13%. Preferred alcohols are selected from the group containingmethanol, ethanol, propanol, or glycerol, ethanol being most preferred.

Optionally, the addition of approximately 1% (v/v) ammonium sulfate tothe mother liquor will generally increase the yield of crystals. Theskilled artisan will also recognize the benefits of adding apreservative such as sodium azide and other such preservatives to themother liquor to prevent bacterial growth.

In another embodiment, mono or disaccharides may be substituted for thealcohol in the same ratios on a weight in volume basis. Mono ordisaccharides suitable for use in the presently claimed process includetrehalose, mannitol, glucose, erythrose, ribose, galactose, fructose,maltose, sucrose, and lactose, though trehalose is preferred.

In yet another embodiment of the present invention, the process may becarried out in a neutral or high pH, zinc-containing environment rangingfrom about pH 7-10, preferably about pH 7.2-9.7. Under these conditions,the GLP concentration is in the range of approximately 1-20 mg/ml,preferably about 2-10 mg/ml. Total zinc, in a molar ratio to GLP, rangesfrom about 0.5-1.7, preferably 0.6-1.5.

Under such neutral or high pH conditions with zinc, suitable buffers andsalts range in concentration from about 10-100 mM glycine and 0-200 mMNaCl, preferably 40-60 mM glycine and 0-150 mM NaCl. Preferred buffersare glycine, aspartic acid and Tris. The alcohol or sugar conditions areas stated previously.

Once the mother liquor is prepared, it is allowed to stand atapproximately 15-37° C., preferably about 18-25° C., for 12-48 hoursuntil crystallization occurs. The crystals may then be transferred orotherwise handled without any noticeable deleterious effects to thecrystalline morphology suggesting that such crystals may be stored forprolonged periods without suffering structural damage.

In another embodiment, a pharmaceutical formulation may be prepared byadding pharmaceutically acceptable excipients, carriers, preservatives,and diluents directly to the mother liquor after the crystals haveformed. In this embodiment, crystallization and subsequent additions areperformed under sterile conditions. Zinc may be added directly to themother liquor to effect the incorporation of zinc into the crystals.Preservatives may be added to the mother liquor to provide formulationsof crystals suitable for multiple injections from the same container.Other excipients, such as antioxidants, buffers, acids and bases for pHadjustments, isotonicity agents and the like, may also be added directlyto the mother liquor after the crystals have formed.

In another embodiment, the invention provides homogenous compositions ofindividual tetragonal flat rod shaped or plate-like crystal of GLP's.Prior to the processes herein disclosed and claimed, such compositionscould not be achieved. The compositions of the invention are useful inmanufacturing processes and for preparing pharmaceutical formulationshaving extended time action for the treatment or prevention of diabetes,obesity and related conditions.

The claimed GLP crystals and compositions may optionally be treated withzinc using conventional crystal soaking techniques. By soaking thecrystals in about a 0.5 mg/ml solution of zinc, complexes of crystalsare formed which serve to extend the time action of the administeredGLP. Also, by varying the zinc concentration, the complex compositioncan be altered leading to longer or shorter time actions.

As noted the invention provides pharmaceutical formulations, which arecomprised of single tetragonal flat rod shaped or plate-like crystal ofa GLP, together with one or more pharmaceutically acceptable diluents,carriers, or excipients. The crystals can be formulated for parenteraladministration for the therapeutic or prophylactic treatment ofdiabetes, obesity or related conditions. For example, the crystals ofthe present invention can be admixed with conventional pharmaceuticalcarriers and excipients. The formulations comprising the claimedcrystals contain from about 0.5 to 50 mg/ml of the active GLP, and morespecifically from about 1.0 to 10 mg/ml. Furthermore, the crystals ofthe present invention may be administered alone or in combination withother antidiabetic agents. For subcutaneous or intramuscularpreparations, a sterile formulation of the crystals of the presentinvention can be administered as a suspension in the original ormodified crystallization mother liquor or in a pharmaceutical diluentsuch as pyrogen-free distilled water, physiological saline, or 5%glucose solution. A suitable formulation of the crystals of the presentinvention may be prepared and administered as a suspension in an aqueousbase or a pharmaceutically acceptable oil base, e.g., an ester of along-chain fatty acid such as ethyl oleate.

Pharmaceutically acceptable preservatives such as an alkylparaben,particularly methylparaben, ethylparaben, propylparaben, or butylparabenor chlorobutanol, phenol or meta-cresol are preferably added to theformulation to allow multi-dose use.

The formulation may also contain an isotonicity agent, which is an agentthat is tolerated physiologically and imparts a suitable toxicity to theformulation to prevent the net flow of water across the cell membrane.Compounds, such as glycerin, are commonly used for such purposes atknown concentrations. Other possible isotonicity agents include salts,e.g., NaCl, dextrose, mannitol, and lactose. Glycerin is the preferredisotonicity agent. The concentration of the isotonicity agent is in therange known in the art for parenteral formulations, and for glycerin, ispreferably about 16 mg/mL to about 25 mg/mL.

The formulation may also contain a pharmaceutically acceptable bufferingagent to control the pH at a desired level. The pH is ideally such as tobe acceptable to the patient upon administration, yet one at which theformulation is sufficiently stable, both physically and chemically.Preferably, the pH is controlled from a mildly acidic pH to a mildlybasic pH, such as, between about pH 5 and pH 9. More preferably, the pHis between about pH 6 and pH 8. Buffering agents include but are notlimited to citrate, acetate, phosphate, Tris, or a basic amino acid,such as, lysine or arginine, which are known to be pharmaceuticallyacceptable in these pH ranges. Other pharmacologically acceptablebuffers for buffering at pH in these ranges are known in the art. Theselection and concentration of buffer is well within the skill of theart.

EXAMPLE 1 pH 6.4 with 1.0% Ammonium Sulfate

12.5 mg of chemically synthesized GLP-1(7-37)OH analog having Valsubstituted for Ala in position 8 (V8-GLP-1) was weighed into a 3.0 mlglass vial and treated with 2.0 ml of 10 mM Tris-HCl, 0.02% NaN₃, pH6.4, to give a clear solution at pH 3.6. The pH of the solution wasadjusted to 8.7 with 2N NaOH and these lowered to pH 6.4 with 1N HCl.The solution remained clear during the pH adjustments. The solution wasfiltered through a 0.22 micron Millex GV13 syringe filter (Millipore,Bedford Mass.) into a new 3.0 ml glass vial. The concentration of theV8-GLP-1 stock solution was 4.76 mg/ml as determined from the absorbanceat 280 mn and using an extinction coefficient of 2.015 for a 1.0 mg/mlV8-GLP-1 solution in a 1 cm cell. A 0.25 ml aliquot of this V8-GLP-1stock solution was transferred to a 2.0 ml glass vial. To this solutionwas added 0.25 ml of a 10 mM Tris-HCl, 0.02% NaN₃, pH 6.4, buffercontaining 2.0% (NH₄)₂SO₄. The vial was sealed, gently swirled, and thenplaced at 18° C. After 36 hours crystalline clusters were identified at200× magnification. For quantitation, a portion of the mother liquor wasremoved and centrifuged at 16,000×g. The V8-GLP-1 content remaining inthe clear supernatant was determined from the absorbance at 280 nm ascited above. The crystalline yield was quantitated by subtracting theV8-GLP-1 level in the supernatant from the V8-GLP-1 level in thestarting solution. This sample showed a crystallization yield of 63.9%.

EXAMPLE 2 pH 6.4 with 1% Ethanol and 1.0% Ammonium Sulfate

A 0.25 ml aliquot of the V8-GLP-1 stock solution was transferred to a2.0 ml glass vial as in Example 1. To this solution was added 0.25 ml ofa 10 mM Tris-HCl, 0.02% NaN₃, pH 6.4, buffer containing 2.0% (NH₄)₂SO₄and 2.0% ethanol. The solution was then treated and evaluated as inExample 1. This sample generated crystalline clusters and a few singletetragonal crystals. The yield was 73.1%.

EXAMPLE 3 pH 6.4 with 5% Ethanol and 1.0% Ammonium Sulfate

A 0.25 ml aliquot of the V8-GLP-1 stock solution was transferred to a2.0 ml glass vial as in Example 1. To this solution was added 0.25 ml ofa 10 mM Tris-HCl, 0.02% NaN₃, pH 6.4, buffer containing 2.0 (NH₄)₂ SO₄and 10.0% ethanol. The solution was then treated and evaluated as inExample 1. This sample generated crystalline clusters, single tetragonalcrystals, and some rods. The yield was 80.3%.

EXAMPLE 4 pH 6.4 with 10% Ethanol and 1.0% Ammonium Sulfate

A 0.25 ml aliquot of the V8-GLP-1 stock solution was transferred to a2.0 ml glass vial as in Example 1. To this solution was added 0.25 ml ofa 10 mM Tris-HCl, 0.02% NaN₃, pH 6.4, buffer containing 2.0% (NH₄)₂SO₄and 20.0% ethanol. The solution was then treated and evaluated as inExample 1. This sample generated single tetragonal crystals and rods.The yield was 81.9%.

EXAMPLE 5 pH 6.4 with 1% Ethanol

A 0.25 ml aliquot of the V8-GLP-1 stock solution was transferred to a2.0 ml glass vial as in Example 1. To this solution was added 0.25 ml ofa 10 mM Tris-HCl, 0.02% NaN₃, pH 6.4, buffer containing 2.0% ethanol.The solution was then treated and evaluated as in Example 1. This samplegenerated a trace of crystal clusters. The yield was 8.8%.

EXAMPLE 6 pH 6.4 with 5% Ethanol

A 0.25 ml aliquot of the V8-GLP-1 stock solution was transferred to a2.0 ml glass vial as in Example 1. To this solution was added 0.25 ml ofa 10 mM Tris-HCl, 0.02% NaN₃, pH 6.4, buffer containing 10.0% ethanol.The solution was then treated and evaluated as in Example 1. This samplegenerated crystal clusters, single tetragonal crystals, and rods. Theyield was 39.1%.

EXAMPLE 7 pH 6.4 with 10% Ethanol

A 0.25 ml aliquot of the V8-GLP-1 stock solution was transferred to a2.0 ml glass vial as in Example 1. To this solution was added 0.25 ml ofa 10 mM Tris-HCl, 0.02% NaN₃, pH 6.4, buffer containing 20.0% ethanol.The solution was then treated and evaluated as in Example 1. This samplegenerated single tetragonal crystals and rods. The yield was 55.5%.

EXAMPLE 8 Pharmacokinetics

28 mg of biosynthetic V8-GLP-1 was weighted into a lass vial anddispersed in 4.5 ml of 10 mM NH₄OAc to give a turbid solution with a pHof 5.6. The material was completely solubilized by adjusting the pH to9.5 with 5N NaOH and remained completely soluble after the pH of thesolution was lowered to 6.4 with 2N HCl. This solution was filteredthrough a 0.22 micron Millex GV13 syringe filter into a new glass vialto give a total volume of 4.3 ml. The concentration of the V8-GLP-1solution was 5.51 mg/ml as determined from the absorbance at 280 nm of a20× dilution of the stock solution and using an extinction coefficientof 2.015 for a 1 mg/ml V8-GLP-1 solution in a 1 cm cell. To thissolution was added 4.3 ml of a 10 mM NH₄OAc, 2.0% (NH₄)₂SO₄, 20%ethanol, pH 6.4, precipitant buffer. The vial was sealed, the solutionwas gently swirled and then placed at 18° C. After 72 hours singletetragonal crystals were identified at 200× magnification. The crystalswere removed from the mother liquor by low speed centrifugation andresuspended in a 10 mM NH₄OAc, 16 mg/ml glycerin, pH 5.5, buffer (bufferA) to a concentration of about 4.0 mg/ml. A portion of the mother liquorwas centrifuged at 16,000×g. The V8-GLP-1 content remaining in the clearsupernatent was determined from the absorbance at 280 nm. Thecrystallization yield was quantitated by subtracting the V8-GLP-1 levelin the supernatant from the V8-GLP-1 level in the starting solution.This crystallization gave an 83% yield.

Calculated aliquots of 4.0 mg/ml V8-GLP-1 crystal suspensions preparedin a similar manner as above were transferred to five glass vials anddiluted with buffer A to a concentration slightly above the final targetconcentration of 2.5 mg/ml V8-GLP-1. To the crystalline suspensions wereadded aliquots of a ZnCl₂ stock solution (33.4 mg/ml Zn⁺⁺in buffer A) tomake final zinc concentrations after 0.5, 1.0, 1.5, or 2.4 mg/ml zinc.The suspensions were gently swirled and placed at 5° C. for 18 hours.The final V8-GLP-1 concentration in each vial was now at the 2.5 mg/mltarget concentration. After 18 hours the crystalline V8-GLP-1 zincsuspensions were transferred to room temperature, passed through a 30gauge needle, and adjusted to pH 6.0 with 1N NaOH.

A 0.1 mg/ml zinc crystalline V8-GLP-1 suspension was prepared by firsttreating a 2.5 mg/ml crystal suspension with 0.15 mg/ml zinc in the samemanner as described above. After 18 hours at 5° C. the zinc treatedcrystals were isolated by low speed centrifugation and transferred tobuffer B (buffer A containing 0.1 mg/ml zinc). The final V8-GLP-1concentration of this suspension was adjusted to the 2.5 mg/ml targetconcentration using buffer B. The suspension was passed through a 30gauge needle, and the pH increased to 6.0 with 1N NaOH.

The give crystalline V8-GLP-1 zinc suspensions descried above, each at2.5 mg/ml V8-GLP-1 and containing 0.1, 0.5, 1.0, 1.5, or 2.4 mg/ml zincwere tested in overnight-fasted beagle dogs. Each animal received asingle 24 nmole/kg subcutaneous injection of the crystalline V8-GLP-1zinc suspension at time zero. Arterial blood samples (1.5 ml) werewithdrawn from the animals at scheduled times, transferred to tubespretreated with EDTA and containing 40 ul of Trasylol, and thencentrifuged. The plasma portion of each sample was separated and storedat −80° C. until analysis. The plasma concentration of immunoreactiveV8-GLP-1 in each sample was measured using a RIA procedure. Table 1shows the resulting immunoreactive V8-GLP-1 plasma levels over a 24 hourtime period for each suspension.

TABLE 1 Immunoreactive V8-GLP-1 Levels (picomolar) in Dog Plasma. 0.1mg/ml 0.5 mg/ml 1.0 mg/ml 1.5 mg/ml 2.4 mg/ml Zinc (n = 5) Zinc (n = 5)Zinc (n = 3) Zinc (n = 5) Zinc (n = 5) Time V8-GLP-1 V8-GLP-1 V8-GLP-1V8-GLP-1 V8-GLP-1 (hrs) (pM) SEM (pM) SEM (pM) SEM (pM) SEM (pM) SEM 0 0  0 0 0 0 0 0 0 0 0 1.5 nd nd 123 41 27 4 7 5 5 5 3.0 nd nd 132 38 387 40 13 27 21 4.5 nd nd 196 51 108 41 76 34 83 37 6.0 301 57 264 79 14055 142 47 143 60 7.5 nd nd 265 71 184 57 198 61 179 63 9.0 nd nd 344 94220 61 252 64 210 66 10.5 nd nd 302 80 231 78 250 66 225 50 12.0 nd nd282 78 236 76 267 60 238 42 13.5 nd nd 238 54 241 97 286 74 236 38 15.0nd nd 263 67 273 118 325 114 246 28 16.5 nd nd 235 51 234 106 275 77 21825 18.0 nd nd 210 47 184 62 254 59 211 23 19.5 nd nd 221 54 209 120 27857 173 9 21.0 nd nd 215 54 219 115 301 48 178 13 22.5 nd nd 224 54 19372 232 23 167 9 24.0 190 30 210 51 187 72 227 34 166 25 30.0 nd nd nd ndnd nd nd nd 171 24 SEM = Standard Error of Mean. nd = not determined

EXAMPLE 9 pH 9.4 with 5% Trehalose and Zinc

6.8 mg of lyophilized biosynthetic V8-GLP-1 was weighed into a 3.0 mlglass vial. Then, 1.0 ml of a 25 mM glycine-HCl, 150 mM NaCl, 5%trehalose, pH 9.0, buffer was added to dissolve the peptide. Thesolution was then adjusted to pH 10.3 with 5N NaOH. While the solutionwas gently stirred 9.0 ul of a 10 mg/ml zinc chloride solution in waterwas added and the pH adjusted to 9.4 with 2N HCl. The finalconcentration of V8-GLP-1 was 5.4 mg/ml as determined from theabsorbance at 280 nm of a 10× dilution of the solution. The solution wasthen filtered with a 0.22 micron Millex GV13 syringe filter. The vialwas capped, gently swirled, and then placed at ambient temperature.After 24 hours V8-GLP-1 crystal clusters and single rectangular crystalswere identified at 430× magnification and estimated to be about 40microns long, 15 microns wide, and 3 microns thick. A portion of themother liquor was removed and centrifuged at 16,000×g. The V8-GLP-1content remaining in the clear supernatant was determined from theabsorbance at 280 nm. The crystalline yield was quantitated bysubtracting the V8-GLP-1 level in the supernatant from the V8-GLP-1level in the starting solution. This sample showed a crystallizationyield of 89.8%. The small rectangular crystal morphology was notobserved in parallel crystallization trials without trehalose.

EXAMPLE 10 pH 9.4 with 10% Mannitol and Zinc

6.8 mg of lyophilized biosynthetic V8-GLP-1 was weighed into a 3.0 mlglass vial, treated with 1.0 ml of a 25 mM glycine-HCl, 150 mM NaCl, 10%mannitol, pH 9.0, buffer and dispersed to give a clear solution. Thesolution was then adjusted to pH 10.3 with 5N NaOH. While the solutionwas gently stirred 9.0 ul of 10 mg/ml zinc chloride solution in waterwas added and the pH adjusted to 9.4 with 2N HCl. The finalconcentration of V8-GLP-1 was 5.31 mg/ml as determined from theabsorbance at 280 nm of a 10× dilution of the crystallization solution.The solution was then filtered with a 0.22 micron Millex GV13 syringefilter. The vial was capped, gently swirled, and then placed at ambienttemperature. After 24 hours, small rectangular plate-like crystals ofV8-GLP-1 were identified at 430× magnification and estimated to be about10 to 30 microns long and 10 microns wide. The yield was determined asin Example 9. This sample showed a crystallization yield of 35%.

EXAMPLE 11 pH 9.0 with Zinc

A 1-ml aliquot of a solution of V8-GLP-1 at 3 mg/ml in 50 mM glycine-150mM NaCl buffer at pH 9.0 was prepared. To this solution was added 7.5 μlof a 20.85 g/ml zinc chloride solution in water, followed by a pHadjustment back up to pH 9.0. After gentle swirling, the clear sample ina 3-ml glass vial was stored at ambient temperature for one day. Afterthis time the crystalline precipitate was examined under the microscopeat 90× magnification, revealing clusters of small plates. Forquantitation of crystallization yield, the entire suspension as passedthrough a 0.2 μm filter (Gelman Sciences, Ann Arbor, Michigan). TheV8-GLP-1 content remaining in the clear filtrate was quantitated byspectroscopic evaluation at a wavelength of 280 nm, using an extinctioncoefficient of 2.015 for a 1 mg/ml solution of V8-GLP-1 in a 1 cm cell.The crystallization yield was quantitated by subtracting the V8-GLP-1level in the supernatant from the V8-GLP-1 level in the startingsolution. This sample showed a crystallization yield of 5.6%.

EXAMPLE 12 pH 9.0 with 10% Ethanol and Zinc

A 1-ml aliquot of a solution of V8-GLP-1 was prepared as in Example 11,except that 110 μl of absolute ethanol was added to the solution priorto the addition of the zinc chloride solution. This sample generatedlarge tetragonal crystals, with some clusters, in 80.6% yield.

EXAMPLE 13 pH 9.5 with 10% Ethanol and Zinc

A solution of V8-GLP-1 at 10 mg/ml in 50 mM glycine-150 mM NaCl bufferat pH 10.5 was passed through a sterile. 0.2 μm Acrodisc filter (GelmanSciences, Ann Arbor, Mich.). To 500 μl of this solution was added 500 μlof a 50 mM glycine-150 mM NaCl buffer at pH 9.0. To this solution wasthen added 110 μl of absolute ethanol followed y 7.5 μl of a 20.85 mg/mlzinc chloride solution in water. Small additions of 1N HCl was used toadjust the solution to pH 9.5. After gentle swirling the final solutionwas enclosed in a 3-ml glass vial and stored at ambient temperature fortwo days. Individual crystalline plates of V8-GLP-1 up to 150 μm inlength, approximately 25 μm wide and less than 5 μm thick were generatedin 72% yield.

EXAMPLE 14 pH 7.9 with 8.5% Ethanol and Zinc

V8-GLP-1 was prepared at 4 mg/ml in 50 mM glycine pH 9.5 buffer,followed by passage through a 0.2 μm filter (Gelman Sciences, Ann Arbor,Michigan). To 1-ml of this solution was added 100 μl of absolute ethanoland then 60 μl of 2.08 mg/ml zinc chloride solution in water. Smalladditions of 0.1N HCl were used to adjust the solution to pH 8.0. Aftergentle swirling the final solution was enclosed in a 3-ml glass vial andstored at ambient temperature for two hours. The pH of the clearsolution was then adjusted to pH 7.86 with small additions of 0.1N HCland storage at ambient temperature continued for two days. Microscopicexamination revealed modest-sized, individual tetragonal plates and someclusters. The V8-GLP-1 content remaining in the clear supernatant wasquantitated by spectroscopic evaluation at a wavelength of 280 nm, usingan extinction coefficient of 2.015 for a 1 mg/ml solution of V8-GLP-1 ina 1 cm cell. The crystallization yield was quantitated by subtractingthe V8-GLP-1 level in the supernatant from the V8-GLP-1 level in thestarting solution. This sample showed a crystallization yield of 92.2%.

EXAMPLE 15 pH 8.3 with 10% Ethanol and Zinc

V8-GLP-1 was prepared at 7 mg/ml in 100 mM glycine pH 10.5 buffer,followed by passage through a 0.2 μm filter (Gelman Sciences, Ann Arbor,Mich.). To 0.5 ml of this solution was added 0.4 ml of water. Then 100μl of absolute ethanol was added, followed by about 6 μl of a 20.86mg/ml zinc chloride solution in water. Small additions of 1N HCl wereused to adjust the solution to pH 8.33. After gentle swirling the finalsolution was enclosed in a 3-ml glass vial and stored at ambienttemperature for one day. Microscopic examination revealed small,individual tetragonal plates and some clusters. The V8-GLP-1 contentremaining in the clear supernatant was quantitated by spectroscopicevaluation at a wavelength of 280 nm, using an extinction coefficient of2.015 for a 1 mg/ml solution of V8-GLP-1 in a 1 cm cell. Thecrystallization yield was quantitated by subtracting the V8-GLP-1 levelin the supernatant from the V8-GLP-1 level in the starting solution.This sample showed a crystallization yield of 92.4%.

EXAMPLE 16 pH 7.4 with 8.6% Ethanol and Zinc

V8-GLP-1 was prepared at 4 mg/ml in 50 mM glycine pH 9.0 buffer,followed by passage through a 0.2 μm filter (Gelman Sciences, Ann Arbor,Mich.). To 5 ml of this solution was added 500 μl of absolute ethanolfollowed by 300 μl of a 2.08 mg/ml zinc chloride solution in water.Small additions of 1N HCl were used to adjust the solution to pH 7.40.After gentle swirling the final solution was enclosed in a 10-ml glassvial and stored at ambient temperature for two days. Microscopicexamination revealed modest-sized, individual tetragonal crystals. TheV8-GLP-1 constant remaining in the clear supernatant was quantitated byspectroscopic evaluation at a wavelength of 280 nm, using an extinctioncoefficient of 2.015 for a 1 mg/ml solution of V8-GLP-1 in a 1 cm cell.The crystallization yield was quantitated by subtracting the V8-GLP-1level in the supernatant from the V8-GLP-1 level in the startingsolution. This sample showed a crystallization yield of 85.0%.

EXAMPLE 17 pH 6.4 with 5% Ethanol and 1.0% Ammonium Sulfate

20 V8-GLP-1 (12.5 mg) was weighed into a 20 ml glass vial. 2.0 ml of a10 mM ammonium acetate buffer containing 150 mM NaCl at pH 6.4 wasadded. The pH of the turbid solution was clarified by adjustment to pH9.5 with 5N NaOH, then lowered to pH 6.4 with 2N HCl. The clear solutionwas filtered through a 0.22 μm Millex GV 13 syringe filter (Millipore,Bedford, Mass.) into a new 20 ml glass vial. The concentration of theV8-GLP-1 stock solution was determined from the absorbance at 280 nmusing an extinction coefficient of 2.015 for a 1.0 mg/ml solution ofV8-GLP-1 in a 1 cm cell. The protein concentration was adjusted to 5.0g/ml. A pH 6.4 precipitant solution containing 10 mM ammonium acetate,150 mM NaCl, 2% ammonium sulfate and 10% ethanol was prepared andfiltered through a 0.22 μm Millex GV13 syringe filter. 2 ml of theprecipitant solution was slowly added to 2 ml of the V8-GLP-1 stocksolution in a glass vial. The vial was gently swirled and incubated atroom temperature for 2 days. Tetragonal plate-shaped crystals wereobserved with a yield of 92%.

The pH of the crystal suspension was adjusted to pH 5.5 with 1N HCl andzinc chloride was added to a final concentration of 0.15 mg/ml. Afterzinc soaking overnight at room temperature, the pH of the suspension wasadjusted to pH 7.5 with 1N NaOH and the preservative meta-cresol wasadded to a concentration of 3.16 mg/ml. This example shows that, ifdesired, preserved formulations of GLP-1 crystals can be prepareddirectly for pharmaceutical use without isolation of the crystals bycentrifugation or filtration in an intermediate step.

EXAMPLE 18 pH 7.6 with 8.5% Ethanol and Zinc

V8-GLP-1 was prepared at 4 mg/ml in 50 mM glycine pH 9.0 buffer,followed by passage through a 0.2 μm filter (Acrodisc from GelmanSciences, Ann Arbor, Mich.). To 10 ml of this solution was added 1 ml ofabsolute ethanol followed by 600 μl of a 6.7 mg/ml zinc acetate(2-hydrate) solution in water. 100 μl of 2% acetic acid was added,resulting in a pH of about 7.6. After gentle swirling the final solutionwas enclosed in a 20-ml glass vial and stored at ambient temperature for24 hours. Microscopic examination revealed modest-sized, individualtetragonal crystals. To the entire solution was then added 3.555 ml ofsolution containing 3.5 ml of a 14 mg/ml solution of m-cresol in waterand 55 μl of 2% acetic acid, resulting in a suspension with a final pHof about 7.2. After gentle swirling the suspension was enclosed in a20-ml glass vial and stored at ambient temperature for 24 hours.Microscopic examination again revealed modest-sized, individualtetragonal crystals.

After centrifugation of an aliquot for 5 minutes at ambient temperature,the V8-GLP-1 content remaining in the clear supernatant was determinedby HPLC analysis of a diluted sample compared to HPLC analysis ofV8-GLP-1 standard solution. The crystallization yield was quantitated bysubtracting the V8-GLP-1 level in the supernatant from the V8-GLP-1level in the starting solution. This preserved v8-GLP-1 formulationshowed a crystallization yield of 97.7%.

EXAMPLE 19 Crystal Stability

Single tetragonal crystals of V8-GLP-1 were prepared in 10 mM NH₄OAc, 1%(NH₄)₂SO₄, 10% ethanol buffer at pH 6.4 at 18° C. as described inExample 8. The crystals were removed from the mother liquor by low speedcentrifugation and resuspended in a 10 mM NH₄OAc, 16 mg/ml glycerin, pH5.5, buffer to a concentration of about 4.9 mg/ml of V8-GLP-1.

2 ml of this suspension was low speed centrifuged and the supernatantwas removed by pipette. The pellet was resuspended in 4 ml of a 10 mMammonium acetate, 16 mg/ml glycerin pH 5.5 buffer containing 0.1 mg/mlzinc. This crystal suspension was allowed to soak in the zinc solutionovernight at 4° C.

The zinc-soaked crystal suspension was divided into four 1-ml aliquots.These suspensions were low speed centrifuged and their supernatants wereremoved by pipette. Four crystal suspensions were prepared in 10 mMammonium acetate, 16 mg/ml glycerin, 0.1 mg/ml zinc buffer at pH 6.0.Further pH adjustments to pH 7.4 with 0.1N NaOH and/or additions ofmeta-cresol to a final concentration of 3.16 mg/ml were made to selectedsamples as illustrated in Table 2. Each suspension was further dividedin half for storage at both room temperature (about 22° C.) and at 4°C., providing a total of 8 test samples as shown in Table 2.

After 10 days, the crystal suspensions were examined under themicroscope. The suspension filtrates were then evaluated by HPLC toquantitate the soluble V8-GLP-1 in the crystalline suspensions. The HPLCresults are reported in Table 2.

TABLE 2 Soluble V8-GLP-1 in Crystal Suspensions after Storage for 10days Storage mg/ml Soluble V8-GLP-1 Sample pH Temperature meta-cresol byHPLC A 6.0  4° C. 0 0.19% B 7.4  4° C. 0 0.10% C 6.0  4° C. 3.16 0.05% D7.4  4° C. 3.16 0.06% E 6.0 22° C. 0 0.16% F 7.4 22° C. 0 0.08% G 6.022° C. 3.16 0.03% H 7.4 22° C. 3.16 0.04%

This experiment showed that less than 0.2% of the 8-GLP-1 peptide becamesolubilized in either the preserved r non-preserved crystal formulationsover a 10-day period.

Microscopically, the crystal suspensions showed less agglomeration orclumping of the single, tetragonal crystals at pH 7.4 than at pH 6.0,and less at 4° C. than at 22° C. The meta-cresol did not seem to have asignificant effect on crystal agglomeration. Additional testing showedthe presence of more than 3% ethanol in the crystal suspension, eitherfrom the original crystallization mother liquor or from subsequentadditions, greatly reduced the clumping tendency of the crystals in bothpreserved and non-preserved formulations. Further tests revealed that,although the crystals are relatively stable in the presence ofmeta-cresol, they are less stable in the presence of 0.5% phenol, whichslowly leads to the formation of amorphous material.

Additional crystal stability tests showed that the V8-GLP-1 crystalsprepared at pH 6.4 are very stable chemically, with no degradation peaksobserved by HPLC analysis after storage at 5° C. or room temperature forup to two months.

Stability tests of crystals prepared in glycine buffer as described inExample 16 showed the V8-GLP-1 crystals stored in the original motherliquor were not stable when meta-cresol was added to the level of 3.16mg/ml. This test resulted in dissolution of the crystals after only 1day. The crystal instability in this composition could be effectivelyblocked by addition of zinc (via a zinc chloride solution) prior toaddition of the preservative.

1. Crystals comprising zinc, and a GLP-1 analog wherein the crystalshave a tetragonal flat rod shaped or plate-like morphology and the GLP-1analog is a peptide of the formula:R₁-X-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Y-Gly-Gln-Ala-Ala-Lys-Z-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-R₂SEQ ID NO:2 wherein: R₁ is selected from the group consisting ofL-histidine, D-histidine, desamino-histidine, 2-amino-histidine,beta-hydroxy-histidine, homohistidine, alpha-fluoromethyl-histidine, andalpha-methyl-histidine; X is selected from the group consisting of Ala,Gly, Val, Thr, Met, Ile, and alpha-methyl-Ala; Y is selected from thegroup consisting of Glu, Gln, Ala, Thr, Ser, and Gly; Z is selected fromthe group consisting of Glu, Gln, Ala, Thr, Ser, and Gly; and R₂ isselected from the group consisting of NH₂, and Gly-OH.
 2. The crystalsof claim 1 wherein X is selected from the group consisting of Gly, Val,Thr, Met, Ile, and alpha-methyl-Ala.
 3. The crystals of claim 2 having asize from between about 2-25 microns by 10-150 microns with a depth fromabout 0.5-5 microns.
 4. A pharmaceutical formulation comprising crystalsas claimed in claim 1 together with one or more pharmaceuticalacceptable diluents, carriers, or excipients.
 5. A pharmaceuticalformulation comprising crystals as claimed in claim 2 with one or morepharmaceutically acceptable diluents, carriers, or excipients.
 6. Apharmaceutical formulation comprising crystals as claimed in claim 3with one or more pharmaceutically acceptable diluents, carriers, orexcipients.
 7. Crystals comprising zinc, and a GLP- 1 analog wherein thecrystals have a tetragonal flat rod shaped or plate-like morphology andthe GLP- 1 analog has at least one modification selected from the groupconsisting of: (a) substitution of glycine, serine, cysteine, threonine,asparagine, glutamine, tyrosine, alanine, valine, isoleucine, leucine,methionine, phenylalanine, arginine, or D-lysine for lysine at position26 and/or position 34; or substitution of glycine, serine, cysteine,threonine, asparagine, glutamine, tyrosine, alanine, valine, isoleucine,leucine, methionine, phenylalanine, lysine, or D-arginine for arginineat position 36; (b) substitution of an oxidation-resistant amino acidfor tryptophan at position 31; (c) substitution of at least one oftyrosine for valine at position 16; lysine for serine at position 18;aspartic acid for glutamic acid at position 21; serine for glycine atposition 22; arginine for glutamine at position 23; arginine for alanineat position 24; and glutamine for lysine at position 26; and (d)substitution comprising at least one of: glycine, serine, or cysteinefor alanine at position 8; aspartic acid, glycine, serine, cysteine,threonine, asparagine, glutamine, tyrosine, alanine, valine, isoleucine,leucine, methionine, or phenylalanine for glutamic acid at position 9;serine, cysteine, threonine, asparagine, glutamine, tyrosine, alanine,valine, isoleucine, leucine, methionine, or phenylalanine for glycine atposition 10; and glutamic acid for aspartic acid at position 15; and (e)substitution of glycine, serine, cysteine, threonine, asparagine,glutamine, tyrosine, alanine, valine, isoleucine, leucine, methionine,or phenylalanine or the D-or N-acylated or alkylated form of histidinefor histidine at position
 7. 8. The crystals of claim 7, wherein theGLP- 1 analog comprises an arginine substituted for lysine at position34.
 9. The crystals of claim 8, wherein an epsilon-amino group of lysineis acylated.
 10. The crystals of claim 7 having a size from betweenabout 2- 25 microns by 10 - 150 microns with a depth from about 0.5 - 5microns.
 11. The crystals of claim 8 having a size from between about 2-25 microns by 10 - 150 microns with a depth from about 0.5 - 5 microns.12. The crystals of claim 9 having a size from between about 2- 25microns by 10 - 150 microns with a depth from about 0.5 - 5 microns. 13.A pharmaceutical formulation comprising crystals as claimed in claim 8with one or more pharmaceutically acceptable diluents, carriers, orexcipients.
 14. A pharmaceutical formulation comprising crystals asclaimed in claim 9 with one or more pharmaceutically acceptablediluents, carriers, or excipients.